protocol illustra card MicroSpin Columns

GE Healthcare

illustraMicroSpin ColumnsFor rapid buffer exchange, desalting and primer removal

Product booklet

Codes: MicroSpin S-200 HR 27-5120-01 (50 purifications)MicroSpin S-300 HR 27-5130-01 (50 purifications)MicroSpin S-400 HR 27-5140-01 (50 purifications)

See back cover for quick reference

protocol card

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Page finder 1. Legal 3

2. Handling 4 2.1. Safety warnings and precautions 4 2.2. Storage 4 2.3. Expiry 4

3. Components 5 3.1. Kit contents 5 3.2. Materials to be supplied by user 5 3.3. Equipment to be supplied by user 5

4. Description 6 4.1. Introduction 6 4.2. The basic principle 7 4.3. Product specifications 9 4.4. When to use an illustra MicroSpin S-200, S-300 or S-400 HR column 9

5. Protocol 12 5.1. Protocol for purification of a range of sample types 13

6. Appendix 15 6.1. RPM calculation from RCF 15 6.2. General guidelines for use of an illustra MicroSpin S-200, S-300 or S-400 HR column 15 6.3. Column specific guidelines for use of illustra MicroSpin S-200, S-300 and S-400 HR columns 17 6.4. Trouble shooting guide 18 6.6. Related products 19

Quick Reference Protocol Card Back Cover Tear off sheet containing a protocol for the experienced user performing buffer exchange, desalting or primer removal

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1. Legal Product use restriction The illustra™ MicroSpin™ S-200, S-300 and S-400 HR Columns and components have been designed, developed and sold for research purposes only. They are suitable for in vitro use only. No claim or representation is intended for their use to identify any specific organism or for clinical use (diagnostic, prognostic, therapeutic, or blood banking).

It is the responsibility of the user to verify the use of the illustra MicroSpin S-200, S-300 and S-400 HR Columns for a specific application, as the performance characteristics of this product have not been verified for any specific organism.

GE, imagination at work and GE monogram are trademarks of General Electric Company.

illustra, GFX, FideliTaq, PuReTaq, Sephacryl, AutoSeq, Hybond, Redivue, Rediprime, NICK, Megaprime, Rapid-Hyb, Hyperfilm, MicroSpin, Hypercassette, ProbeQuant, NAP and Ready-To-Go are trademarks of GE Healthcare companies

All third party trademarks are the property of their respective owners.

© 2006–2010 General Electric Company-All rights reserved. Previously published July 2006.

All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available upon request. Contact your GE Healthcare representative for the most current information (see back cover for contact details).

http://www.gelifesciences.com

GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire. HP7 9NA UK.

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2. Handling

2.2. Storage All kit components should be stored at 4°C. Do not freeze.

2.3. Expiry For expiry date please refer to outer packaging label.

2.1. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.

All chemicals should be considered as potentially hazardous. Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products. Suitable protective clothing such as laboratory overalls, safety glasses and gloves should be worn. Care should be taken to avoid contact with skin or eyes; if contact should occur, wash immediately with water (See Material Safety Data Sheet(s) and/or Safety Statement(s) for specific recommendations).

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3. Components

3.1. Kit contents Identification Pack size

Cat. No.50 purifications

27-5120-01, 27-5130-01 or 27-5140-01

illustra™ MicroSpin™ S-200, S-300 and S-400

HR columns

50

Collection tubes 50

Refer to the Certificate of Analysis for a complete list of kit components.

3.2. Materials to be supplied by user Disposables: 1.5 ml DNase-free microcentrifuge tubes.

3.3. Equipment to be supplied by user Microcentrifuge that accommodates 1.5 ml microcentrifuge tubesVortex mixer (optional)

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4. Description

4.1. Introductionillustra MicroSpin S-200, S-300 and S-400 HR columns contain Sephacryl™ resin of differing pore sizes. They allow DNA purification by the process of gel filtration. Molecules larger than the largest pores in the Sephacryl are excluded from the gel and elute first. Intermediate size molecules penetrate the matrix to varying extents, depending on their size and the resin used. Penetration of the matrix retards progress through the column; very small molecules elute last. The volume required to elute these small molecules is dependent on the volume available both inside and outside the pores i.e. the bed volume.

Gel filtration resins do not exhibit a fixed exclusion limit when used in a spin-column format. Exclusion limits of gel filtration resins are only meaningful in continuous flow processes where the molecules being purified have sufficient time to reach equilibrium between the time spent in the gel filtration medium and the time spent in the eluent stream. In spin-column chromatography, the observed exclusion properties that allow the product to pass through the gel while the smaller impurities are retained depends on experimental factors, such as: the resin used, sample volume, product size, and the g forces used in the purification process.

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4.2. The basic principle

Column Preparation

Sample Application

Elution

1.

Use of illustra MicroSpin S-200, S-300 and S-400 HR columns involves the following steps:

3.

2.

Purified DNA ready for downstream applications

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Step Comments Component

1. Column Preparation

The resin is re-suspended and excess storage

buffer removed by centrifugation.

illustra MicroSpin S-200, S-300 and S-400 HR column

2. Sample Application

The sample is applied to the column.

3. Elution Purified sample is eluted by

centrifugation.

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4.3. Product specifications

Table 1. illustra MicroSpin S-200, S-300 and S400 HR column specifications

Sample Type: DNA radiolabeling reaction

Principle Gel filtration

Column matrix Sephacryl resin of appropriate pore size

Column storage buffer

TE buffer (10 mM Tris/HCl, 1mM EDTA, pH 7.6)

Input sample volume 25–100 μl

Percent sample recovery

> 80%

Length of labeled DNA recovered

Variable-depends on input sample

Nuclease Testing: Column components are tested in nickase, single and double-stranded exonuclease

and RNase assays.

Major subsequent applications

Dependent on input sample, but includes cloning, PCR, blotting and sequencing

applications

4.4. When to use an illustra MicroSpin S-200, S-300 or S-400 HR columnillustra MicroSpin S-200, S-300 and S-400 HR columns are designed for the rapid purification of DNA for a wide range of applications, including desalting, buffer exchange, removal of dye terminators from cycle sequencing reactions and removal of labeled nucleotides from DNA labeling reactions. Good product yield and purity is obtained with sample volumes from 25–100 μl, and from

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Application Product Product code

Pack size

PCR reaction and enyzymatic DNA reaction purification 50 bp–10 kbp size range Extraction of DNA from agarose gels

illustra GFX™ PCR DNA and

Gel Band Purification Kit

28-9034-70

28-9034-71

100 purifications

250 purifications

Dye terminator removal from automated sequencing reactions

illustra AutoSeq G-50

27-5340-01

27-5340-02

27-5340-03

50 purifications

50 purifications

50 purifications

Unincorporated labeled nucleotide removal from a DNA labeling reaction (> 20 mers)

illustra ProbeQuant G-50 Micro-

Columns

28-9034-08 50 purifications

nanogram to milligram quantities of DNA. Enzymes will not be denatured or removed. For guidelines to consider for use of illustra MicroSpin S-200, S-300 and S-400 HR Columns, please see sections 6.2 & 6.3.

GE Healthcare provides a wide range of nucleic acid purification products, some of which may be better suited to your application. These products and the application for which they have been optimized are summarized in Table 1 below. illustra AutoSeq™ G-50 Columns and illustra ProbeQuant™ G-50 Micro Columns are provided pre-equilibrated in the optimal buffer for the application for which they are designed


Pack size

Purification of oligonucleotides following synthesis, buffer exchange and de-salting. Gravity format, 500 μl loading volume

illustra NAP™-5 Columns


Spin column format 150 μl loading volume

illustra MicroSpin

G-25 Columns


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Note: Columns are NOT transferable between GE Healthcare kits, e.g., the composition of the MicroSpin S-200, S-300 and S-400 HR Columns is not the same as the composition of the MicroSpin G-50 Columns.

Use of icons The Key below describes the purpose of the icons used throughout the protocol booklet.

This icon is used to highlight particularly critical steps within the protocol that must be adhered to. If this advice is not followed it will have a detrimental impact on results.

This icon is used to highlight technical tips that will enhance the description of the step. These tips may indicate areas of flexibility in the protocol or give a recommendation to obtain optimum performance of the kit.

See section 6.2 & 6.3 for processing and analysis of these samples.

See section 3.2 and 3.3 for Materials & Equipment to be supplied by user.

5. Protocol

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5.1. Protocol for purification of a range of sample types1. Column Preparation a. Re-suspend the resin in the column by

vortexing.

b. Loosen the cap one-quarter turn and twist off the bottom closure.

c. Place the column in the supplied Collection tube for support.

d. Spin for 1 minute at 735 × g.

Note: See section 6.1 for RPM calculation from RCF.

Note: Use columns immediately after preparation to avoid drying out of the resin. If the column resin appears dry, displaced or cracked after the first spin, this is usually indicative of over-centrifugation (too fast or too long). Re-hydrate the column with 250 μl of TE buffer, vortex and re-centrifuge, checking the settings. Spin speed can be reduced by 20% if necessary. Do not use the pulse button on the microcentrifuge as this may over-ride the speed setting.

e. Proceed immediately to step 2 below.

2. Sample Application a. Place the column into a fresh DNase-free 1.5 ml

microcentrifuge tube (user supplied).

1 minute735 × g

b. Slowly apply 25–100 μl sample to the top-center of the resin, being careful not to disturb the resin bed (see section 6.2 for volume of sample to add).

Note: The resin will have come away from the column slightly to form a pillar. It is essential that the sample being purified is applied slowly and is not allowed to run down the sides of the resin bed. Avoid touching the resin bed with the pipet tip.

3. Elutiona. Spin for 2 minutes at 735 × g.

The purified sample is collected in the bottom of the 1.5 ml microcentrifuge tube.

b. Cap the microcentrifuge tube.

c. Store the purified probe at -20°C.

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25–100 μl sample

2 minutes735 × g

6. Appendices

6.1. RPM calculation from RCF The appropriate centrifugation speed for a specific rotor can be calculated from the following formula:

RPM = 1 000 × √(RCF/1.12r)

Where RCF = relative centrifugal force, r = radius in mm measured from the center of the spindle to the bottom of the rotor bucket, and RPM = revolutions per minute.

For example, if an RCF of 735 × g is required using a rotor with a radius of 73 mm, the corresponding RPM would be 3 000.

Table 1 below shows appropriate RPM for various microcentrifuges.

Table 1: Appropriate RPM for an RCF of 735 × g

Microcentrifuge Appropriate RPM for an RCF of 735 × g

Heraeus Biofuge 15 2 800 Beckman GS15R 2 100 Hettich Mikro 24-48 2 630 Hettich Mikro EBA12 2 700 Eppendorf Centrifuge 5415C 3 000 Eppendorf Centrifuge 5417C 2 700

6.2. General guidelines for use of illustra MicroSpin S-200, S-300 and S-400 HR columns illustra MicroSpin S-200, S-300 and S-400 HR columns can be used for a wide variety of DNA purification applications. When using these columns, consider the following guidelines:

20× rule The best results will be obtained when the product being purified is at least 20 times larger than the largest impurity. If the

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difference in size is less than 20-fold, either purity or yield may be compromised.

Purity versus yield In general, purity is inversely proportional to yield. Larger sample volumes will provide higher yield but lower purity, and vice-versa. For any given volume, the larger the pore size of the resin, the greater the purity and lower the yield of the product which results. Gel filtration matrices with larger pore sizes (Sephacryl S-400>Sephacryl S-300>Sephacryl S-200) tend to retain more product than gel matrices with smaller pores.

Non-specific binding The non-specific binding exhibited by the illustra MicroSpin S-200, S-300 and S-400 HR Columns is relatively insignificant, allowing purification of samples in the nanogram range. There will be a uniform proportional loss of sample which is due to the nature of spin column chromatography.

Retention For a given sample volume, product retention is relative to molecular size. As the size of the product increases, its relative retention decreases.

Loading volumes Load 25–100 μl onto a column for all applications.

For larger sample volumes, either use more than one column or reduce the sample volume by drying or precipitation. For smaller sample volumes, dilute the sample to improve product recovery. If the volume recommendations are followed, the yield of purified DNA is expected to be 50–90%.

Enzyme Removal For purification of DNA fragments 50 bp–10 kbp in length, following an enzymatic reaction, we recommend using the illustra GFX PCR DNA and Gel Band Purification Kit, as the enzyme will be removed during the spin column process. If using an illustra MicroSpin S-200, S-300, or S-400 HR column, you must Phenol Chloroform extract prior to loading onto the column to ensure enzyme removal.

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6.3. Column specific guidelines for use of illustra MicroSpin S-200, S-300 and S-400 HR Columns For more specific column selection, a simplified applications guide is given in Table 3 below. Table 3: Column specific guidelines

Application Notes Recommended Column

Reaction volume

PCR reaction and enyzymatic DNA reaction purification (buffer exchange, de-salting)

Will not remove

enzyme*

S-200 25–50 μl

PCR reaction and enyzymatic DNA reaction purification (removal of excess primers prior to cloning)

Will not remove

enzyme*

S-400 25–50 μl

PCR reaction and enyzymatic DNA reaction purification (removal of excess primers prior to other applications)

Will not remove

enzyme*

S-300 S-400

25–50 μl 50–100 μl

Unincorporated labeled nucleotide removal from a DNA labeling reaction**

S-200 S-300 S-400

25–50 μl 50–75 μl

75–100 μl

*To remove enzyme from PCR or enzymatic reactions, use illustra GFX PCR DNA and Gel Band Purification Kit or Phenol Chloroform extract sample.** DNA must be at least 100 bp in length for a good recovery. For DNA less than 100 bp in length, use illustra ProbeQuant G-50 Micro Columns.

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For removal of unincorporated nucleotides from oligonucleotides, use illustra MicroSpin G-25 Columns.

Exceptions exist to these guides:

• Use illustra MicroSpin S-300 HR Columns for removal of primers from PCR reactions < 200 bp in length, regardless of the intended application.

• Use illustra MicroSpin S-400 HR Columns to remove primers that are greater than 24 bases in length, regardless of the size of PCR product.

6.4. Troubleshooting guideThis guide may be helpful in the first instance, however if problems persist or for further information please contact GE Healthcare technical services. Telephone numbers are on the back page. Alternatively log onto http://www.gelifesciences.com/illustra.

Problem: Resin appears dry and cracked after Column Preparation step.

Possible causes Suggestions

Poor sample purity • Ensure the sample volume was within acceptable range prior to loading (see section 6.2).

• Ensure sample is CAREFULLY pipetted into center of resin. Do not disturb the column. Do not allow the sample to run into the sides of the resin bed.

• Use the column immediately after completing Column Preparation step. Do not allow the resin to become dried out or cracked.

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6 .6. Related productsA full range of Molecular Biology reagents can be found in the GE Healthcare catalog and on the web site http://www.gelifesciences.com/illustra

A full range of Detection Products and available pack sizes can be found in the GE Healthcare catalog and on the web site http://www.gelifesciences.com/newhyperfilm

If you need further information, GE technical services are happy to assist (world-wide phone numbers can be found on the back cover).


Pack size

Blotting Hybond™-N+ (82 mm)

RPN82B 50 discs

Hybond-N+ (15 × 20 cm)

RPN1520B 10 sheets

Hybond-NX (82 mm)

RPN82T 50 discs

Hybond-NX (15 × 20 cm)

RPN1520T 10 sheets

Hybond-N (82 mm)

RPN82N 50 discs

Hybond-N (15 × 20 cm)

RPN1520N 10 sheets

Hybond-XL (82 mm)

RPN82S 50 discs

Hybond-XL (15 × 20 cm)

RPN1520S 10 sheets

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Pack size

Blotting Continued

Hybond blotting paper

(20 × 20 cm)

RPN6101M 100 sheets

Radioactive labeling

Rediprime™ II DNA Labeling System

RPN1633 30 reactions

Ready-To-Go™ DNA Labeling Beads (-dCTP)

27-9240-01 1 kit

Megaprime™ DNA Labeling System,

dNTP


Megaprime DNA Labeling System,

dCTP


Nick Translation Kit, dNTP

N5500 20 reactions

Nick Translation Kit, dCTP

N5000 20 reactions

5’-End Labeling Kit RPN1509 20 reactions

Rapid-Hyb™ Buffer RPN1635 125 ml

Detection Hyperfilm™ MP (18 × 24 cm)

28-9068-43 50 sheets

Hyperfilm MP Enveloped

(18 × 24 cm)

28-9068-50 50 sheets

Hypercassette™ RPN11642 1

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Pack size

Purification of DNA probes

illustra MicroSpin G-25 Columns


and oligonucleotides

illustra ProbeQuant G-50 Micro Columns


illustra NICK™ Columns


illustra NAP-5 Columns


Purification of DNA from PCR, agarose

illustra GFX PCR DNA & Gel Band Purification Kit


gel bands and enzymes

illustra GFX 96 PCR Purification Kit

28-9034-45 10 × 96 well plates

Preparation of PCR products for automated sequencing

ExoSAP-IT™ US78200 100 reactions

Dye terminator removal from automated sequencing reactions

illustra AutoSeq G-50 Columns


Kits containing ready-to-use mix for PCR amplification

illustra Hot Start Master Mix

25-1500-01 100 reactions

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Pack size

Kits containing

ready-to-use mix

for PCR

illustra PuReTaq™ Ready-To-Go PCR

Beads

27-9557-01 96 reactions in 0.2 ml

tubes/plate

amplification

Continuedillustra PuReTaq

Ready-To-Go PCR Beads

27-9557-02 5 × 96 reactions in

0.2 ml tubes/plate

FideliTaq™ PCR Master Mix Plus

(2 ×)

E71182 100 reactions

FideliTaq Master Mix Plus

E71183 100 reactions

Premixed nucleotides for PCR amplification

illustra DNA Polymerization Mix dNTP Set (A,C,G,T)

20 mM each

28-4065-57 10 μmol

illustra DNA Polymerization Mix dNTP Set (A,C,G,T)

20 mM each

28-4065-58 40 μmol (4 × 10 μmol)

illustra PCR Nucleotide Mix

dNTP Set (A,C,G,T) 25 mM each

28-4065-60 500 μmol

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Pack size

Premixed nucleotides for PCR amplification Continued

illustra PCR Nucleotide Mix

dNTP Set (A,C,G,T) 2 mM each

28-4065-62 1 ml

27-5120-01PL AF 03/2010

For your local office contact information, visit http://www.gelifesciences.com/contact

GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK

http://www.gelifesciences.com

imagination at work

GE Healthcare offices:

GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala Sweden

GE Healthcare Europe GmbH Munzinger Strasse 5 D-79111 Freiburg Germany

GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK

GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway NJ 08855-1327 USA

GE Healthcare Japan Corporation Sanken Bldg. 3-25-1 Hyakunincho Shinjuku-ku Tokyo 169-0073 Japan

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protocol illustra card MicroSpin Columns

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Transcript of protocol illustra card MicroSpin Columns